A Systematic Study on the Optimal Nucleotide Analogue Concentration and Rate Limiting Nucleotide of the SARS-CoV-2 RNA-Dependent RNA Polymerase

Figures

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  Figure 1. Inhibitory effect of GS-443902 and cordycepin-TP on the SARS-CoV-2 RdRp complex. (A) Schematic of the polymerase reaction. A 40-mer RNA oligonucleotide was annealed with a 20-mer, radioactive label on its 5′ end. The green arrow indicates the incorporation position 1 of an adenosine or adenosine analog. The red arrow indicates position +3, after the first adenosine incorporation position. (B) Chemical structure of the used triphosphates GC-443902 and cordycepin-TP and their corresponding ProTide structures GS-5734 and NUC-7738. (C) Radiogram of the polymerase reaction. The drug concentrations are increasing from 500 nM, 5 μM, 50 μM to 500 μM. Lane 1: positive control reaction without inhibitor and 10 μM NTP concentration. Lanes 2–5: increasing concentrations of GS-443902, stalling position of the RdRp complex is indicated with a red arrow. Lane 6: positive control without inhibitor, indicating inefficient polymerization under ΔATP conditions, compared to the full NTP pool as shown as in lane 1. Lanes 7–10: ATP depleted reaction with increasing GS-443902 concentrations, stalling position is indicated with a red arrow. Lanes 7–9 show increasing amounts of full-length product…

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  Figure 2. IC50 determination for GS-443902 and cordycepin-TP. (A) The polymerization assay with a pool of 0.5 μM NTP, under increasing amounts of GS-443902 from 0μM to 81.9 μM. The assay was used to determine the IC50 concentration of GS-443902 for 0.5 μM and 1 μM competing NTP concentrations. (B) Same assay as in (A) but with cordycepin-TP as inhibitor. (C) The assay was used to calculate the IC50 for competing NTP concentrations of 1 μM and 0.5 μM for GS-443902. (D) IC50 determination for competing NTP concentrations of 1 μM, 0.5 μM and 0.1 μM for cordycepin-TP. When compared to GS-443902, cordycepin-TP reaches a 100% inhibition, while GS-443902 still leads to the full-length product. The level of 100% activity was determined by the 40-mer signal in the control reaction without inhibitor (lane 1) and 0% activity by the absence of the 40-mer signal. The radiographs for GC-443902 and Cordycepin-TP were conducted in, respectively, one and two replicates per NTP concentration.

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  Figure 3. Effect of NTP depletion–SARS-CoV-2 RdRp is sensitive to GTP depletion. (A) The polymerase reaction (as described in Figure 1A) was performed under decreasing concentrations of NTPs at 1 mM, 100 μM, 10 μM, and 1 μM. For ΔUTP, the range was 1 mM, 10 μM and 1 μM. Subsequently the NTP pool was depleted for each NTP individually. A negative control in lane 1 contained the heat-denatured protein complex, indicating the 20-mer that is not elongated. Lanes 2–5 indicate decreasing concentration of a pool of all four NTPs. Lanes 6–9, ΔGTP, are an NTP pool lacking GTP and stalling at position 12 (blue arrow). In lanes 10–13, the ΔATP condition is shown, indicating stalling at 1 μM NTP concentration at position 2 (brown arrow). Lanes 14–17, ΔCTP, indicate stalling at position 8 (magenta arrow). Lanes 18–20 lack UTP and stall at position 0 at 1 μM NTP concentration (yellow arrow). (B) Polymerase reaction indicating the stalling position. This radiograph is a representative imagine of two independent experiments.